PURPOSE: The present experiments were undertaken to evaluate the validity of absolute flow velocity measurements with a dual-beam bidirectional Doppler Fourier-domain optical coherence tomography (FD-OCT) system.METHODS: The flow velocities of diluted milk through a glass capillary were measured at 30 different preset velocities in the range of 0.9 to 39.3 mm/s by bidirectional Doppler FD-OCT. The flow through the capillary was controlled by two infusion pumps working in different flow ranges and based on different technical principles. In vivo the validity of the method for measuring blood flow in retinal vessels was tested at bifurcations. The continuity equation was verified at 10 retinal venous bifurcations of 10 young healthy subjects (mean age, 29 ± 3 years) by velocity measurements, using dual-beam bidirectional Doppler FD-OCT, and measurements of retinal diameters, using the Dynamic Vessel Analyzer.RESULTS: Flow velocities as measured with bidirectional Doppler FD-OCT in the glass capillary were in good agreement with the preset velocities (r = 0.994, P < 0.001 each). No significant difference was found between flow in the trunk vessels before the bifurcation (11.3 ± 5.2 μL/min) and the sum of flows in the daughter vessels (10.7 ± 4.8 μL/min). A significant association was found between retinal vessel diameters and both retinal blood velocities (r = 0.72, P < 0.001) and retinal blood flow (r = 0.95, P < 0.0001).CONCLUSIONS: Dual-beam bidirectional Doppler FD-OCT delivered accurate retinal blood velocity values and, thus, offers high potential for examination of retinal blood flow in ocular disease.